Turk J Med Sci

2010; 40 (5): 729-734

Original Article TBTAK
E-mail: medsci@tubitak.gov.tr
doi:10.3906/sag-0812-9

Adrenergic and cholinergic responses of the tracheal smooth

muscle is enhanced during estrus phase in rat

M. Kasm AYCI1, Yasemin AYDIN2, Kubilay UZUNER2

Aim: The aim of this study was to evaluate influences of hormonal changes during rat estrous cycle on airway
responsiveness to bronchoconstrictor and bronchodilator agents.
Materials and methods: The study was performed in 5 groups of rat tracheal smooth muscle obtained during proestrus,
estrus, metestrus, and diestrus phases of the estrous cycle, as well as the ovariectomized group (n:8/group). Cumulative
acetylcholine contractions ranging from 10-8 to 10-3 M bath doses and the cumulative isoproterenol relaxations ranging
from 10-8 to 10-3 M precontracted with 10-5 M of acetylcholine were recorded on isolated tracheas placed in a
computerized tissue bath system.
Results: Acetylcholine-induced contractions ranging from 10-5 to 10-3 M were significantly enhanced dose-dependently
in tracheal smooth muscle from estrus rats compared to those from metestrus and ovariectomized rats. The relaxation
-8 -3
induced by isoproterenol (10 -10 M) also significantly increased in tracheal smooth muscle precontracted with
-5
acetylcholine (10 M) taken from estrus rats. However, acetylcholine-induced contractility increase in tracheal smooth
muscle was much more prominent than isoproterenol-induced relaxations in tracheal smooth muscle from estrus rats.
Conclusion: These findings indicated that changes in estrogen and/or progesterone levels during rat estrous cycle might
affect acetylcholine and isoproterenol sensitivity of the tracheal smooth muscle.

Key words: Acetylcholine, estradiol, estrous cycle, isoproterenol, tracheal smooth muscle

Sanda estrus faz srasnda trakea dz kas adrenerjik ve kolinerjik cevaplar artar
Ama: Bu almann amac san estrus dngs srasndaki hormonal deiikliklerin, bronkokonstriktr ve
bronkodilatr ajanlara, solunum yolu hassasiyeti zerine etkilerinin deerlendirilmesidir.
Yntem ve gere: alma estrus dngsnn proestrus, estrus, metestrus ve diestrus fazlarndaki ve ovarektomi
uygulanm sanlar olmak zere 5 grup sandan (n:8/grup) elde edilen trakea dz kasnda yaplmtr. zole trakealar
bilgisayarl organ banyosu sistemine yerletirilip banyo dozlar 10-8 den 10-3 Ma kadar olan kmlatif asetilkolin
-5
kaslmalar ve 10 M asetilkolinle kastrlm prekontrakte preparatlarda ayn dozajlardaki kmlatif izoproterenol
gevemeleri kaydedilmitir.
Bulgular: Asetilkolinle (10-8-10-3 M) uyarlm kaslmalar estrustaki sanlarda metestrustaki ve ovarektomi uygulanm
sanlarla karlatrldnda doza bal olarak anlaml bir ekilde artmtr. Estrustaki sanlardan alnm olan ve
-8 -3
asetilkolinle nceden kastrlm trakea dz kaslarnda izoproterenolle (10 -10 M) uyarlm gevemeler de anlaml bir
ekilde artmtr. Ancak estrustaki sanlardan alnan trakea dz kasndaki asetilkolinle uyarlan kaslabilirlikteki art
izoproterenolle uyarlm gevemeden daha belirgindir.
Sonu: Bu bulgular san estrus dngs srasndaki estrojen ve/veya progesteron seviyelerindeki deiikliklerin trakea
dz kasnn asetilkolin ve izoproterenole hassasiyetini etkilediini gstermektedir.

Anahtar szckler: Asetilkolin, estradiol, estrus dngs, izoproterenol, trakea dz kas

Received: 01.12.2008 Accepted: 13.04.2010

1
Department of Biology, Faculty of Science and Arts, Dumlupnar University, Ktahya - TURKEY
2
Department of Physiology, Faculty of Medicine, Eskisehir Osmangazi University, Eskiehir - TURKEY
Correspondence: M. Kasm AYCI, Department of Biology, Faculty of Science and Arts, Dumlupnar University, Ktahya - TURKEY
E-mail: kcayci@dumlupinar.edu.tr

729
Estrous cycle alters tracheal smooth muscle responses
Introduction
Hormonal changes during menstrual cycle in
human and estrous cycle in animals may differentially
influence physiologic and pathophysiological
responses of body systems (1). Autonomic nerve
function may be modulated in various ways, such as
neurotransmitters, inflammatory mediators, or
hormones. There are some reports emphasizing the
complexity of interactions of factors that regulate
smooth muscle response to adrenergic and
cholinergic response differences depending on the
phase of the estrous cycle (2-4). Receptor density of
neurotransmitters in various target tissues is
modulated by sex hormones, as well.
Evidence from several studies suggests a role for
sex hormones in the pathogenesis of asthma. Among
the general population, asthma prevalence is higher
in women than in men (5). Female reproductive
processes, such as pregnancy and menstruation, affect
asthma, indicating a major role for the female sex
hormones. Studies have suggested that respiratory
function is influenced by female sex hormones and
menstrual cycle phase (6,7). The lung has hormone
receptors for both estrogen and progesterone (8-10).
Gonzalez-Arenas et al. (11) reported that during the
estrous cycle, various progesterone and estrogen
receptors have been found at rat lung, among which
progesterone receptor isoforms are the highest during
proestrus and the lowest during estrus.
Since the sex steroid changes during the menstrual
cycle differentially affect the smooth muscle
sensitivity to cholinergic and adrenergic agents,
changes in the tracheal smooth muscle sensitivity
depending on the phase of estrous cycle may have
important implications on the asthma profile of
pregnant women and female patients, especially those
who are given high doses of estrogen replacement
therapy. However, observations regarding the
influence of estrogens on asthma are conflicting. In a
cohort study, long-term use and/or high doses of
postmenopausal estrogen therapy have been reported
to increase the subsequent risk of asthma (12). In
contrast, others have suggested that estrogen
treatment can have a beneficial effect on airway
responsiveness (13) and on asthma (14). Degano et al.
730
(15) have shown that low physiologic doses of 17-
estradiol given to ovariectomized female rats
decreased airway responsiveness to acetylcholine.
Effects of estrogen on whether muscarinic or
adrenergic sensitivity of rat isolated tracheal smooth
muscle is not clear. Because of this, in this study, we
aimed to investigate effects of phases of rat sexual
cycle on rat tracheal smooth muscle contraction to
acetylcholine and relaxation to isoproterenol.
Materials and methods
Animals
Adult female Sprague-Dawley rats weighing 200-
250 g obtained from animal house of our department
were used for the study. Rats were housed under
temperature-controlled room with a 12 h light: 12 h
dark cycle (lights on at 7:00 am), and fed with
standard pellet and water ad libitum. Animal studies
were performed with the approval of Animal Care and
Ethics Committee of Medical Faculty of Eskisehir
Osmangazi University.
Determination of estrous cycle
Estrous cycle phases were determined by daily
vaginal smears every morning at 9:00 as described by
Marcondes et al. (16) according to the following
criteria: a proestrus smear consisting of a
predominance of nucleated epithelial cells; an estrus
smear primarily consisting of anucleated cornified
cells; a metestrus smear consisting of the same
proportion among leukocytes and cornified and
nucleated epithelial cells; and a diestrus smear
primarily consisting of a predominance of leukocytes.
Only those rats that had at least 2 regular 4-day
estrous cycles were used for the experiments.
Ovariectomy
In one group, rats were bilaterally ovariectomized.
Ovariectomies were performed under aseptic
conditions. Animals were anesthetized with a mixture
of xylazine hydrochloride (10 mg/kg, i.p.) and
ketamine hydrochloride (25 mg/ kg, i.p.). Dorsolateral
incisions were made. The ovary and oviduct and a
small section of the uterus were removed.
Ovariectomies were performed at least 4 weeks before
organ-bath studies.
 M. K. AYCI, Y. AYDIN, K. UZUNER

Organ-bath studies
After determination of the estrous cycle phase, the
rats were sacrificed by cervical dislocation. Trachea
between larynx and bifurcation was dissected out
quickly and the connective tissues were gently
removed. The trachea was cut into helical fashion in
a cold oxygenated Krebs-Henseleit solution (pH 7.40)
containing (in mM) NaCl (118), KCl (5.4), MgSO4
(1.2), KH2PO4 (1.2), NaHCO3 (25.0), glucose (11.7),
and CaCl2 (2.5). Each preparation was vertically
mounted in a 10 mL organ-bath containing Krebs-
Henseleit solution (pH 7.4, 37 C) continuously
bubbled with 95% O2 and 5% CO2. The lower hooks
were anchored to the bottom of the baths, and the
upper hooks were attached to a force displacement
transducer (Biopac, USA) connected to a data
acquisition and analysis system (MP 100, Biopac,
USA).
The preparations were allowed to equilibrate for 1
h under 1 g initial tension. During the equilibration
period, the bath solution was changed every 15 min.
After the equilibrium period, the trachea was tested
with 80 mM KCl to evaluate the viability of the tissue.
The preparations were exposed to acetylcholine
(10-8-10-3 M, Sigma-Aldrich, USA) or isoproterenol
(10-8-10-3 M, Sigma-Aldrich, USA). Concentrations
were not increased until the response to the previous
concentration had stabilized. For isoproterenol-
induced relaxation studies following the equilibration
502.5 124 mg) were significantly greater than those
in metestrus (maximal contraction; 331.8 25 mg)
and ovariectomized (maximal contraction; 355.4
113 mg) groups (P < 0.05) (Figure 1).
Isoproterenol-induced relaxations in the trachea
precontracted with acetylcholine were significantly
enhanced in estrus (maximal relaxation; 112.1 15
mg) when compared with proestrus (maximal
relaxation; 46.3 10 mg) and diestrus (maximal
relaxation; 72.9 23 mg) as illustrated in Figure 2 (P
< 0.05).
Contraction sensitivity was much more prominent
than relaxation sensitivity of tracheal smooth muscle
in estrus; EC50 of acetylcholine-induced contraction
increased to 236.20 59 mg, but EC50 of
isoproterenol-induced relaxation was only 72.65
9.25 mg.
Discussion
Involvement of estrogens on asthma development
has not been clearly defined yet. Estrogen treatments
600
proestrus
500 estrus
metestrus
diestrus
Contraction (mg)

400 ovariectomized
period, trachea was precontracted with 10-5 M *
acetylcholine. 300 *
*
Statistical analysis *
200
All data were expressed as mean SD. Statistical
*
differences of the results were determined by one-way 100
analysis of variance followed by a Tukey test. *
0
Statistical significance was considered as P < 0.05. 9 8 7 6 5 4 3
Ach, -log M

Results Figure 1. Cumulative contraction response to acetylcholine in

Acetylcholine treatment to tracheal preparations
produced dose dependent contraction as shown in
Figure 1. With 10-5 M to 10-3 M doses, acetylcholine-
induced contractions in estrus (maximal contraction;
tracheal smooth muscle from different phases of rat
estrous cycle: proestrus (n = 8), estrus (n = 8),
metestrus (n = 7), diestrus (n = 8), and ovariectomized
(n = 8). Data were expressed as mean SD of absolute
force (mg) values. *P < 0.05 compared with the estrus
data.

731
Estrous cycle alters tracheal smooth muscle responses
Isoproterenol, -log M
9 8 7 6 5 4
0 of 17-estradiol elevate uterus nuclear estrogen
*
-20 * stimulate protein synthesis in hours. Klangkalya et al.
*
* *
Relaxation (mg)
-40
* *
-60
-80
proestrus
estrus
-100 metestrus
diestrus
ovariectomized
-120
Figure 2. Cumulative relaxation response to isoproterenol in
tracheal smooth muscle precontracted with
acetylcholine (105 M) from different phases of rat
estrous cycle: proestrus (n = 8), estrus (n = 8),
metestrus (n = 7), diestrus (n = 8), and ovariectomized
(n = 8). Data were expressed as mean SD of absolute
force (mg) values. *P < 0.05 compared with the estrus
data.
have been reported either to be beneficial on airway
responsiveness (12) or to increase the risk of asthma
(13,14). The present study suggested that changing sex
steroid profile during estrous cycle modulates
cholinergic and adrenergic responses of isolated rat
tracheal smooth muscle.
At estrous cycle, estrogen is high prior to the
ovulation and is low following the ovulation (17-19).
Herein we show that cholinergic contraction of
tracheal smooth muscle is greater at high estrogen
levels than at low estrogen levels. Consistent with this
observation, Degano et al. (20) reported that in
ovariectomized female rats, chronic treatment with
17-estradiol could alter airway reactivity in a dose-
dependent manner: low dose estradiol treatment
decreases and high dose estradiol treatment increases
the potency of acetylcholine.
In the present study, we showed that cholinergic
contraction and adrenergic relaxation of tracheal
smooth muscle were highest in estrus. Consistent with
this observation, Honda et al. (3) reported that
acetylcholine-induced relaxation of the thoracic aorta
was greatest in estrus.
732
Medlock et al. (21) showed that physiologic doses
3
receptors within 1-3 h; indicating that estrogens
(4) reported that cardiac muscarinic and beta-
adrenergic receptor density increased upon treatment
* with progesterone and estrogen together in
* ovariectomized rats. Moreover, Levin et al. (22)
reported that density of muscarinic and adrenergic
receptors increased in rabbit bladder given 4-days of
estradiol treatment. Additionally, Wilkinson et al. (23)
reported that estrogen induces an increase in
sensitivity of alpha-receptors and numbers of beta-
receptors in rat hypothalamus. Thus, we suggested
that one of the reasons why both acetylcholine
induced contraction and isoproterenol induced
adrenergic relaxation were highest in estrus that
following proestrus may be the result of time
dependent genomic effects of the elevated estrogen
during proestrus. In other words, the effects of the
elevated estrogen in proestrus may change
acetylcholine and isoproterenol sensitive receptor
activity and density of the tracheal smooth muscle of
the rat.
Our study suggested that the lowest contraction to
acetylcholine was seen in metestrus and
ovariectomized rats that were expected to have the
lowest estrogen concentration, which further supports
the idea that increased estrogen may affect
acetylcholine and isoproterenol sensitive receptor
activity of tracheal smooth muscle. Additionally, the
presence of high progesterone that hyperpolarizes
smooth muscles during metestrus may also be
involved in the relaxation response and the
suppressed contraction response. On the other hand,
there are 2 progesterone peaks: one in metestrus and
the other in proestrus (17,19). The elevated
progesterone during proestrus may counteract on the
acetylcholine induced contractions in this phase, as
well.
Considering the reports cited above and our
results, increasing acetylcholine-induced contraction
and isoproterenol-induced relaxation in tracheal
smooth muscle during estrus phase may be due to; i.
the increase in production of estrogens and/or

progesterone dependent bronchodilators and
bronchoconstrictors, ii. changes in adrenergic and/or
cholinergic receptor sensitivity and density, iii.
changes in adrenergic and cholinergic metabolizing
systems, iv. direct-indirect potentiation of adrenergic
and cholinergic responses, and v. estrogen induced
modulation of intracellular Ca2+ mobilization.
In conclusion, our data support the idea that
changes in steroid profile during estrous cycle alter
airway smooth muscle contraction and relaxation
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